Aromatic compounds synthesized by the polyketide route are probably the most commonly occurring phenolic derivatives of natural origin, with the exception of the lignins. There is however almost no information available on their metabolic turnover or the mechanisms used by micro- organisms to metabolize these compounds. This program proposes to: (A) establish the main types of catabolic routes used by micro-organisms to degrade these compounds; (B) study in detail the oxygenases involved in these transformations; (C) establish the relationships, if any, of the enzymes (or metabolic sequences) involved, to the well described pathways of metabolism of other aromatic compounds; (D) provide further evidence for non-specific reaction sequences being responsible for the nutritional diversity of some micro-organisms; (E) substantiate the general hypothesis that acquisition of new enzyme function occurs by the alteration of substrate specificity of existing enzymes. Micro-organisms, capable of metabolizing naturally occurring and synthetic compounds, have been isolated, and the pathways of resorcinol and orcinol (3,5-dihydroxytoluene) metabolism have been partially elucidated. The first enzyme of the reaction sequences resorcinol and orcinol hydroxylases are flavoproteins one of which we have crystallized. We propose to study these hydroxylases and the m- hydroxybenzoate hydroxylases (also flavoproteins) from other pseudomonads in detail. Comparisons will be made of their physical properties and subunit structure, catalytic potential by study of the partial reactions, kinetics and specificity (three substrates are involved in these reactions), stereospecificity, NIH shift, the alterations in substrate specificity induced by mutation and immunological homologies. The techniques used will include polarography, spectrophotometry, including the use of stopped flow devices, fluorimetry, and ORD-CD for the studies of the kinetic and catalytic properties of the enzyme. Well controlled chemostats will be used to apply continuous selective pressures for the evolution of mutants.